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Gβγ mediates activation of Rho guanine nucleotide exchange factor ARHGEF17 that promotes metastatic lung cancer progression

2021; Elsevier BV; Volume: 298; Issue: 1 Linguagem: Inglês

10.1016/j.jbc.2021.101440

1083-351X

Irving García‐Jiménez, Rodolfo Daniel Cervantes‐Villagrana, Jorge Eduardo del-Río-Robles, Alejandro Castillo-Kauil, Yarely Mabell Beltrán‐Navarro, Jonathan García-Román, Guadalupe Reyes‐Cruz, José Vázquez‐Prado,

Erythrocyte Function and Pathophysiology

Metastatic lung cancer is a major cause of death worldwide. Dissemination of cancer cells can be facilitated by various agonists within the tumor microenvironment, including by lysophosphatidic acid (LPA). We postulate that Rho guanine nucleotide exchange factors (RhoGEFs), which integrate signaling cues driving cell migration, are critical effectors in metastatic cancer. Specifically, we addressed the hypothetical role of ARHGEF17, a RhoGEF, as a potential effector of Gβγ in metastatic lung cancer cells responding to LPA. Here, we show that ARHGEF17, originally identified as a tumor endothelial marker, is involved in tumor growth and metastatic dissemination of lung cancer cells in an immunocompetent murine model. Gene expression–based analysis of lung cancer datasets showed that increased levels of ARHGEF17 correlated with reduced survival of patients with advanced-stage tumors. Cellular assays also revealed that this RhoGEF participates in the invasive and migratory responses elicited by Gi protein–coupled LPA receptors via the Gβγ subunit complex. We demonstrate that this signaling heterodimer promoted ARHGEF17 recruitment to the cell periphery and actin fibers. Moreover, Gβγ allosterically activates ARHGEF17 by the removal of inhibitory intramolecular restrictions. Taken together, our results indicate that ARHGEF17 may be a valid potential target in the treatment of metastatic lung cancer. Metastatic lung cancer is a major cause of death worldwide. Dissemination of cancer cells can be facilitated by various agonists within the tumor microenvironment, including by lysophosphatidic acid (LPA). We postulate that Rho guanine nucleotide exchange factors (RhoGEFs), which integrate signaling cues driving cell migration, are critical effectors in metastatic cancer. Specifically, we addressed the hypothetical role of ARHGEF17, a RhoGEF, as a potential effector of Gβγ in metastatic lung cancer cells responding to LPA. Here, we show that ARHGEF17, originally identified as a tumor endothelial marker, is involved in tumor growth and metastatic dissemination of lung cancer cells in an immunocompetent murine model. Gene expression–based analysis of lung cancer datasets showed that increased levels of ARHGEF17 correlated with reduced survival of patients with advanced-stage tumors. Cellular assays also revealed that this RhoGEF participates in the invasive and migratory responses elicited by Gi protein–coupled LPA receptors via the Gβγ subunit complex. We demonstrate that this signaling heterodimer promoted ARHGEF17 recruitment to the cell periphery and actin fibers. Moreover, Gβγ allosterically activates ARHGEF17 by the removal of inhibitory intramolecular restrictions. 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Cell Biol. 2016; 212: 647-659Crossref PubMed Scopus (16) Google Scholar). Since cell migration and proper control of the cell cycle are altered in cancer cells, the finding that ARHGEF17 is mechanistically linked to these essential processes is consistent with a putative dysregulation of this RhoGEF in carcinogenesis and metastasis. Although ARHGEF17 was originally identified as a tumor endothelial transcript (51St Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Genes expressed in human tumor endothelium.Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1638) Google Scholar), its potential role in cancer progression remains unknown. Since increased expression of ARHGEF17 correlated with bad prognosis in lung cancer patients with high-grade tumors, here we analyzed its role in tumor growth and metastasis. We used immunocompetent mice as a preclinical model to address the involvement of ARHGEF17 in tumorigenesis by syngeneic LAP0297 lung cancer cells and established the mechanism by which Gi-coupled LPARs activate this RhoGEF. To get an initial insight into the potential role of ARHGEF17 in lung cancer we looked for clinical differences in patients having this gene amplified compared with all others. Analysis of the non–small cell lung carcinoma (NSCLC) TCGA datasets (61Campbell J.D. Alexandrov A. Kim J. Wala J. Berger A.H. Pedamallu C.S. Shukla S.A. Guo G. Brooks A.N. Murray B.A. Imielinski M. Hu X. Ling S. Akbani R. Rosenberg M. et al.Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas.Nat. Genet. 2016; 48: 607-616Crossref PubMed Scopus (682) Google Scholar) revealed that ARHGEF17 amplification correlated with a higher percentage of patients with tumors at stages II–IV and N1–2, indicative of advanced tumor growth and lymph node infiltration, respectively (https://www.cbioportal.org/; Fig. 1, A and B; ARHGEF17 is named GEF17 in all the figures). Consistent with these observations, maximally separated Kaplan–Meier survival plots of patients with high-grade tumors (stage II–IV) had a significant statistical correlation between high ARHGEF17 expression and reduced survival (Fig. 1C). In contrast, patients with tumors classified as stage I did not show statistical correlation between high ARHGEF17 expression and overall survival (not shown). These analyses were consistent with a hypothetical role of ARHGEF17 in lung cancer progression. To directly address the potential role of ARHGEF17 in metastatic lung cancer, we knocked down this RhoGEF in LAP0297 lung cancer cells using a lentiviral shRNA-ARHGEF17 (Fig. 1D) to address their tumorigenic and organotropic metastatic potential in immunocompetent FVB mice. LAP0297 shGEF17 cells, with reduced expression of ARHGEF17, developed smaller tumors compared with sh-Control cells (Fig. 1E), which, at the end of the experiment, weighted significantly less (Fig. 1, F and G). The metastatic potential of LAP0297-shGEF17 knockdown lung cancer cells, compared with sh-Control cells, was evaluated in FVB mice that were intravenously injected in the tail with 500,000 cells. Metastatic tumors grown in the lungs were evaluated after 2 weeks. The macroscopic appearance of normal lungs and those excised from mice injected with sh-Control and shGEF17 cells is shown in Figure 1H. Lungs with multiple metastatic tumors weighted more than normal lungs from healthy mice (Fig. 1H). Interestingly, both the weight (Fig. 1H) and the number of lung macrometastasis (Fig. 1I) were significantly attenuated in mice inoculated with cells in which ARHGEF17 was knocked down in comparison to those inoculated with sh-Control cells. The putative antiproliferative effect of ARHGEF17 knockdown and the potential effect on cell viability were assayed in sh-Control and shGEF17-cultured LAP0297 cells. For these experiments, cells were incubated overnight with 1% fetal bovine serum (FBS) and then incubated with 10% FBS or serum-free media for 48 h and analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. ARHGEF17 knockdown shGEF17 cells were able to proliferate in response to 10% FBS (Fig. 1J), and their viability was not significantly reduced in the prolonged absence of serum (Fig. 1K). However, when compared with sh-Control cells, the proliferative effect was slightly reduced (Fig. 1J) as well as the viability in the continued absence of serum (Fig. 1K). Together, these results suggest that ARHGEF17 plays a critical role in lung cancer growth and metastatic dissemination. We previously demonstrated that LPA stimulates metastatic LAP0297 cells to migrate via a signaling pathway sensitive to pertussis toxin (PTX) and gallein, indicating the critical participation of heterotrimeric Gi proteins controlling a still to be revealed migratory mechanism based on Gβγ-effector pathways (50Cervantes-Villagrana R.D. Color-Aparicio V.M. Reyes-Cruz G. Vazquez-Prado J. Protumoral bone marrow-derived cells migrate via Gbetagamma-dependent signaling pathways and exhibit a complex repertoire of RhoGEFs.J. Cell Commun. Signal. 2019; 13: 179-191Crossref PubMed Scopus (5) Google Scholar). Consistent with the importance of LPA signaling in lung cancer (41Magkrioti C. Oikonomou N. Kaffe E. Mouratis M.A. Xylourgidis N. Barbayianni I. Megadoukas P. Harokopos V. Valavanis C. Chun J. Kosma A. Stathopoulos G.T. Bouros E. Bouros D. Syrigos K. et al.The autotaxin-lysophosphatidic acid axis promotes lung carcinogenesis.Cancer Res. 2018; 78: 3634-3644Crossref PubMed Scopus (35) Google Scholar), analysis of transcriptomic information from the NSCLC TCGA datasets revealed a positive correlation between ARHGEF17 and autotaxin (ectonucleotide pyrophosphatase/phosphodiesterase 2 [ENPP2]) expre